Exogenous application of nanocarrier‐mediated double‐stranded RNA manipulates physiological traits and defence response against bacterial diseases

Abstract Stability and delivery are major challenges associated with exogenous double‐stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly‐aspartic acid‐derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP‐overexpressing transgenic plants was significantly reduced after infiltration with gfp‐dsRNA‐CPP6 by silencing of the transgene compared to plants treated only with gfp‐dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft‐dsRNA‐CPP6 and pif4‐dsRNA‐CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds‐dsRNA‐CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl‐induced OsbZIP23 was targeted through root uptake of bzip23‐dsRNA‐CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer‐encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.


| INTRODUC TI ON
Many abiotic and biotic factors such as drought, temperature, insects, bacteria, fungi and viruses limit plant growth, development and yield (Kamthan et al., 2015).Several omics and functional studies have been performed to identify the factors that limit plant adaptation to stress and productivity (Mittler & Blumwald, 2010;Tester & Langridge, 2010).Many small molecules, plant growth regulators and pesticides to counter these limitations have been identified, although their functions may be reversible and they may require repeated applications to achieve the desired efficacy (Margaritopoulos et al., 2020;Shah & Shad, 2020).Genetic manipulation of plant traits adversely affected by abiotic and biotic stresses have emerged as potential targets for crop improvement.As there are strong biosafety regulations and limited public acceptance of genetically modified (GM) crops, the development of transgene-free plants is desirable but is also laborious and time-consuming (Lusser et al., 2012).In this context, instantaneous and easy-to-execute strategies are needed.RNAi technology has been used for functional studies of genes and double-stranded RNA (dsRNA) application has emerged as a highly promising technique to target genes to manipulate plant processes and improve crop protection against insects, fungi and viruses (Ibrahim et al., 2011;Simón-Mateo & García, 2011;Xin et al., 2010).DICER-LIKE proteins process the dsRNA inside the cells to produce small interfering RNAs (siRNAs), which are recognized by Argonaute (AGO) proteins to form RNAinduced silencing complexes (RISC) (Vergani-Junior et al., 2021).RISC along with siRNAs base pair with complementary mRNA to cleave the transcript or inhibit its translation (Fire et al., 1998).RNAi-based technologies are considered low risk and could reduce the usage of chemical pesticides to attain sustainability goals.
The topical application of dsRNA can control plant viruses such as pepper mottle virus, tobacco mosaic virus and bean common mosaic virus (Konakalla et al., 2016;Mitter, Worrall, Robinson, Xu, et al., 2017;Tenllado et al., 2003;Worrall et al., 2019).MoDES1, a host-defence suppressor pathogenicity gene of Magnaporthe oryzae that has been targeted by application of exogenous dsRNA, confers resistance against fungal blast disease in rice (Sarkar & Roy-Barman, 2021).Host-induced gene silencing (HIGS) strategies have been effectively used against pathogens and pests such as viruses, insects, fungi and nematodes (Koch & Wassenegger, 2021).
The success of HIGS depends on the efficiency of plant DCL proteins involved in processing siRNAs, the uptake ability of siRNAs by the pathogen and feeding behaviour of animal vectors (Wang et al., 2015).HIGS vectors with hairpin sequences have been used to express dsRNAs in plants.dsRNA targeting the CarE gene of Sitobion avenae expressed in wheat delayed larval growth (Xu et al., 2014).
Transgenic plants expressing dsRNA (CYP3-RNA) targeting all three copies of the Fusarium graminearum CYP51 gene (FgCYP51A,FgCYP51B,FgCYP51C) in Arabidopsis and barley (Hordeum vulgare) inhibited fungal infection through HIGS (Koch et al., 2019;Nowara et al., 2010).The success of HIGS leads to the direct delivery of siRNAs against pathogen/insect genes using environment-friendly spray-induced gene silencing (SIGS) approaches.The exogenous application of dsRNAs targeting S. avenae structural sheath protein (Shp), involved in aphid feeding behaviour, protected barley plants (Biedenkopf et al., 2020).Targeting M. oryzae MoDES1, an innate defence suppressor of rice, using SIGS confers partial resistance to the fungus (Sarkar & Roy-Barman, 2021).Although dsRNAs have been used to target virus, insect and fungal genes, attempts to target plant endogenous genes and bacterial genes are limited.RNAi technologies have successfully improved crop yield by targeting agronomically important traits like height, branching and increased biomass.
Knockdown of OsDWARF4 in rice caused improved yield as plants possess short height and erect leaf architecture with increased photosynthesis in lower leaves (Feldmann, 2006).Maize Corngrass1 (Cg1) miRNA overexpression in Arabidopsis and switchgrass prolongs the vegetative phase, delays flowering, increases biomass with 250% more starch and improved digestibility (Chuck et al., 2011).
In rice, bacterial leaf blight (BLB) disease is caused by Xanthomonas oryzae pv.oryzae (Xoo) can severely affect yield.Streptomycin and copper oxychloride-based pesticides have been used for crop protection; however, prolonged usage of pesticides leads to the development of resistant strains (Prasad et al., 2018).Therefore, there is a demand for alternative ways to control pathogenicity and to improve agronomic traits.Several genes may be targeted to manipulate plant growth and improve resistance against Xoo (Bakade et al., 2021;Pal et al., 2022;Vemanna et al., 2019).Overexpression of transcription factor OsWRKY62 compromised the basal defence and Xa21-mediated resistance to Xoo (Peng et al., 2008).OsWAK12 is a negative regulator for rice blast disease that altering basal resistance (Delteil et al., 2016).Xoo secretes transcription-activator-like effectors (TALEs) into plants that bind to the promoter of SWEET14 (Bezrutczyk et al., 2018;Oliva et al., 2019;White et al., 2009).
Arabidopsis plants overexpressing salt-and drought-induced RING box (SDIR1) are susceptible to Pseudomonas syringae pv.tomato (Pst) and sdir1 mutants are resistant (Ramu, Oh, et al., 2021;Ramu, Pal, et al., 2021).Targeting such negative regulators using dsRNA is likely to provide an opportunity to manipulate plant growth, immunity and productivity.However, the major challenge that remains is the stability and regulated delivery of the dsRNA to the site of action, because naked dsRNA is highly prone to degradation in the natural environment due to conditions like sunlight, UV light and pH (Christiaens et al., 2020).Therefore, attempts have been made to deliver dsRNA using nanoparticles.Delivery of dsRNA with clay nanosheets provides sustained protection against cucumber mosaic or pepper mild mottle viruses in local and systemic tissues (Mitter, Worrall, Robinson, Li, et al., 2017).The foliar application of dsRNA conjugated with bioclay effectively protected plants against the phloem-feeding pest Bemisia tabaci (Mitter, Worrall, Robinson, Li, et al., 2017).The dsRNA is processed into 21-22 nucleotide (nt) siR-NAs by whitefly RNAi machinery, which leads to homologous transcript degradation and thus insect mortality (Jain et al., 2022).
One challenge in HIGS is the systemic spread of the activated RNAi machinery.To achieve the maximum effectiveness of gene silencing, robust nanopolymers with systemic movement ability are required to deliver dsRNA.We report that cationic poly-aspartic-derived

| Nanoformulation-based topical delivery of dsRNA into plants
Naked dsRNA delivery into plants is challenging in natural environmental conditions as it is highly prone to degradation by UV light, microorganisms and washout in rain.A range of polymeric nanocarriers having structural flexibility for modulation, bioconjugation, degradability and stability in varied biological systems were utilized to deliver dsRNA.Five different cationic poly-aspartic acid-derived polymers (CPP1, CPP2, CPP4, CPP6, and CPP8), biocompatible and biodegradable with varied hydrophobicity, were synthesized previously (Figure 1a) and can effectively deliver nucleic acids into mammalian cells (Yavvari et al., 2019).No inhibitory effect on shoot and root length by these different CPPs on rice seedlings at 5, 25, 50 and 75 μg/mL concentrations were observed (Figure S1).The CPPs did not interfere with the growth and development of the seedlings.The CPP6-based formulation was previously reported to be the most efficient for intracellular delivery of siRNAs against the SUMOylation machinery of the mammalian mouse model to reduce gut inflammation.CPP6 can form polyplexes with siRNA and maintained moderate positive zeta potential (ZP) with the highest transfection efficiency in the mammalian system (Yavvari et al., 2019).Thus, we chose CPP6 to deliver dsRNAs into plant cells.dsRNA targeting GFP was used to test the mass ratio required to stabilize nucleic acids in the complex.
CPP6 conjugated with 500 ng of dsRNA was incubated in a 1:1, 1:5, 1:10, 1:25 and 1:50 wt/wt ratio at room temperature for 30 min and resolved on an agarose gel.Even at 1:1 ratio, the polymer could encapsulate dsRNA quite effectively (Figure 1b).The layered double hydroxide (LDH) polymer efficiency was also assessed at 1:1, 1:5, 1:10, 1:25 and 1:50 wt/wt ratio and showed similar encapsulation of dsRNA (Figure 1c).The transfection efficiency of polymers in delivering siR-NAs in the mammalian systems is effective with a 1:10 ratio (Yavvari et al., 2019).At lower ratios, the dsRNA may be released quickly and not efficiently silence the plant's endogenous genes.Therefore, a 1:10 ratio of dsRNA:CPP6 was chosen for dsRNA delivery and durable effectiveness in plants to silence the target genes.A hydrodynamic diameter (D H ) size range of 200-500 nm was measured for CPP6 with gfp-dsRNA and pds-dsRNA (Figure 1d,e).Similarly, the ZP of CPP6 with dsRNA of gfp and pds was >10 mV (Figure 1f).To assess the release of dsRNA from CPP complexes, different concentrations of sodium dodecyl sulphate (SDS) were used; 0.1% SDS could completely release the dsRNA (Figure 1g).Higher concentrations of SDS formed micelles and interacted with ethidium bromide, giving fluorescent anionic bands that migrated down the agarose gel, as reported by Holt et al. (2020).The success of exogenous application of dsRNA on plants depends on systemic translocation and prolonged availability of dsRNA to activate RNAi machinery.The stability of dsRNA-CPP6 complex and naked dsRNA was assessed by incubation at 4, 37 and 48°C for 10 days.Naked dsRNA at 48°C showed degradation after 4 days and was not detectable after 10 days, whereas the dsRNA-CPP6 complex showed negligible release of dsRNA from the polymer (Figures 1h and S2a).Similarly, the stability of the dsRNA-CPP6 complex at pH 7, 5 and 3 was assessed.Naked dsRNA was degraded at pH 3 after 10 days whereas the dsRNA-CPP6 complex showed no release of dsRNA, suggesting the efficiency of CPP6 in nucleic acid protection (Figures 1i and S2b).The stability of dsRNA-CPP6 complex and naked dsRNA was also assessed by incubating with RNase A at 4, 37 and 48°C (Figures 1j and S2c,d).RNase A treatment did not influence either the release or degradation of dsRNA.
CPP6 and LDH polymer complexes provide stability to dsRNA, and so we investigated whether these polymers could interfere with DNA template accessibility for polymerase activity in replication, transcription and translation, which is also useful for gene silencing.PCRs containing the GFP template, CPP6 and gfp-dsRNA-CPP6 complexes produced no amplified fragments.In contrast, naked gfp-dsRNA did not inhibit the reaction, suggesting that CPP6 interfered with template DNA accessibility for the amplification (Figures 1k and S2d).
However, with LDH polymer at 100 ng and 200 ng, PCR amplification of the genes was unaffected, suggesting their inefficiency in interfering with GFP template accessibility (Figure 1l).To assess whether CPP6 and LDH polymers interfere with luciferase (luc)-dsRNA and inhibit the translation process, an coupled transcription and translation assay was performed.CPP6, at 500 ng in a coupled transcription/translation reaction in vitro, reduced the relative luminescence activity of luciferase, whereas LDH polymer, even at 2000 ng, showed higher luminescence compared to the control reaction.The luc-dsRNA-CPP6 complex showed a significant reduction in relative luminescence compared to the control, whereas naked dsRNA showed some extent of reduction (Figure 1m).This suggests that CPP6 can interfere in the suppression of genes during the translation process and demonstrates that dsRNA-CPP6 affects gene silencing at multiple levels.
To test CPP6 efficiency in plant cellular uptake and systemic spread to different plant parts, Cy7-labelled CPP6 was infiltrated into Nicotiana benthamiana and Arabidopsis thaliana leaves.Fluorescence was detected in leaves after 24 and 48 h (Figure 2a

| Silencing of GFP in Arabidopsis using dsRNA-CPP6
To test the efficiency of dsRNA in silencing plant endogenous genes, transgenic Arabidopsis plants overexpressing the green fluorescent protein gene (GFP-OE) were developed.Naked gfp-dsRNA and gfp-dsRNA-CPP6 complex were syringe-infiltrated into 3-week-old GFP-OE plants (Figure 3a).GFP transcript levels were reduced in  plants showed a reduction of protein levels compared to the GFP-OE control plants, confirming the systemic movement and extended availability of dsRNA due to CPP6 (Figure 3d).

| Foliar application of dsRNA-CPP6 can regulate flowering genes in Arabidopsis
The major focus of our study was to target the plant endogenous genes to modulate plant processes related to growth, development, stress and disease resistance in a transient manner.FT and PIF4 in Arabidopsis were targeted by respective dsRNA-CPP6, which caused a delay in flowering time and enhanced biomass.FT is known to translocate through the phloem from leaves to the shoot apical meristem, causing a switch from vegetative to reproductive organ development.FT overexpression in various species accelerates flowering.PIF4, a bHLH transcription factor, regulates FT activation through direct binding to its promoter, and RNAi lines have delayed flowering (Kumar et al., 2012).To delay flowering and improve biomass in Arabidopsis, FT and PIF4 transcripts were targeted through dsRNA (Figure 4a).Arabidopsis Col-0 plants, before bolting, were exogenously sprayed with 125 ng/plant ft-dsRNA and ft-dsRNA-CPP6.At Expression of another predicted off-target gene Pectin methyl esterase inhibitor (PMEI) was also reduced in ft-dsRNA-CPP6-treated plants (Figure 4i).The higher expression of PMEI in Arabidopsis inhibits floral organ primordia (Peaucelle et al., 2008).
The upstream regulator of FT, PIF4, was targeted by pif4-dsRNA.

| dsRNA uptake in rice seedlings can effectively silence genes
To assess the effectiveness of dsRNA-mediated silencing in rice, we targeted the phytoene desaturase (PDS) gene using seedling dipinoculation.Three-day-old seedlings were treated with 2 μg of naked pds-dsRNA or pds-dsRNA-CPP6 and allowed to take it up through the roots.At 10 days post-treatment (dpt), pds-dsRNA-treated seedlings showed reduced growth with a yellowish to albino phenotype (Figure 5a).The seedling height was reduced >1-fold in pds-dsRNA-CPP6-treated samples compared to CPP6-alone treated and control seedlings (Figure 5b).PDS transcript levels did not show any reduction compared to control plants.However, a typical albino phenotype was observed (Figure 5c).OsPDS is required at higher levels during early-stage development, thus transcript levels were not reduced; however, pds-dsRNA-CPP6-sprayed plants showed an albino phenotype and reduced growth.We hypothesize that stress-induced transcripts of negative regulators may be efficiently targeted through dsRNA.OsbZIP23 transcription factor is upregulated in salt, drought and oxidative stress conditions (Pa et al., 2022;Sujitha et al., 2023).
OsbZIP23 was therefore targeted by dsRNA.The germinated rice seedlings were exposed to NaCl-induced stress and treated with bzip23-dsRNA-CPP6.At 48 hpt, the growth of seedlings in NaCl was reduced; however, the growth of plants treated with naked bzip23-dsRNA was not affected (Figure 5d,e).The expression of bZIP23 under NaCl stress was triggered significantly compared to the control, and in polymer-alone or bzip23-dsRNA-alone treated seedlings, no induction was observed.However, in NaCl-exposed seedlings treated with bzip23-dsRNA-CPP6, the expression of bZIP23 was significantly reduced, suggesting the effective silencing of the target gene (Figure 5f).The reduction in transcript levels also affected the expression of the bZIP23 target gene, Overlay Tolerant to Salt 1 (OTS1), which positively correlated under NaCl treatment (Figure 5g).
To assess the effectiveness of dsRNA-CPP6 on Arabidopsis infected with Pst, bacterial culture containing sdir1-dsRNA or sdir1-dsRNA-CPP6 was syringe infiltrated.Arabidopsis plants treated with dsRNA targeted to AtSDIR1 showed reduced transcript levels, bacterial growth (Figure 6a,b) and a healthy phenotype (Figure S4a).The predicted off-target DEK domain-containing chromatin-associated protein-encoding transcripts were unaltered in all plants (Figure 6c).

| DISCUSS ION
We have demonstrated the efficiency of CPP6 as a nanocarrier to deliver dsRNA through foliar spray to induce gene silencing (SIGS), manipulating plant endogenous genes for crop protection and improvement.The success of gene silencing in field conditions can only be achieved by spraying the dsRNA targeting relevant genes.The major challenge in SIGS is dsRNA stability, delivery and systemic spread for target gene silencing (Li et al., 2015).Another challenge in exogenous applications is the cell wall barrier to deliver nucleic acids into plant cells.These challenges forced us to apply nanomaterials in transfecting nucleic acids for crop improvement.In plants, the negative charge of dsRNA prevents passive transport through negatively charged membranes (Wytinck et al., 2020).To deliver siRNAs, abrasion and high-pressure sprays have been used to improve the cellular uptake in plants (Dalakouras et al., 2016).siRNAs are known to move cell-to-cell (short range) and systemically (long range) movement through plasmodesmata and vascular phloem tissue (Melnyk  et al., 2011).The delivery of dsRNA is highly dependent on their absorption into cells.Many cationic cell-penetrating peptides and lipid carriers have been used for gene delivery (Milletti, 2012).Various nanomaterials like carbon nanotubes, magnetic nanoparticles, mesoporous silica nanoparticles and LDH nanoparticles have been reported to deliver nucleic acids into plant cells (Bao et al., 2016;Kolge et al., 2021;Mitter, Worrall, Robinson, Li, et al., 2017).CPP effectively encapsulated the dsRNA at a 1:1 ratio and was stable at various pH and temperatures.CPP6 could systemically spread from the site of infection, as evidenced by NIR signals in distal leaves and GFP silencing in transgenic Arabidopsis plants.CPP6 was also taken up through roots and spread systemically to the shoots in rice, which could help deliver a wide variety of agronomically important molecules other than nucleic acids.dsRNA can also enter plant cells through the foliar spray and spread systemically to shoots, leaves and roots, probably through phloem.However, the exact mechanism behind the uptake and translocation remains unclear.CPP is highly soluble in water, and simple detergent-like compounds in plant cells can easily release the conjugated dsRNA effectively.Poly-aspartic acid is biodegradable (Adelnia et al., 2019) and thus, CPP6 may not pose any environmental threat to use in agricultural fields.The efficient binding of CPPs to nucleic acids can suppress enzymatic degradation.The CPP6 binding to the template RNA inhibited translation, resulting in effective gene silencing.
Large-scale production and application of dsRNAs and their adaptation in farming could enhance agricultural productivity, reduce malnutrition and sustain food security by manipulating relevant genes.Targeting specific genes may improve basic agronomic traits, grain yield, fruit quality and enhance shelf-life.
The dsRNA-CPP6 targeting FT and PIF4 delayed flowering, which resulted in more leaves and increased biomass.The reduced expression of predicted off-target genes MgT and PMEI is mainly due to a delay in flowering, where signals for floral transition did not induce the expression of these genes.In Arabidopsis and rice, overexpression of miR156 targeting SQUAMOSA (SQUA) promoter-binding-like (SPL) gene, delays flowering and increases biomass (Schwab et al., 2005;Xie et al., 2006).The transcription factor PIF4 directly binds to the promoter of AtFT and induces flowering (Kumar et al., 2012).Silencing of SlPIF4 in tomatoes causes stunted growth, 15% reduction in vegetative weight and 23% reduction in fruit weight with 21% total reduction in biomass (Rosado et al., 2019).
The dsRNA applications to modify the plant metabolic process could be very attractive to alter plant growth and development.
RNAi hairpin construct can modulate the blue flower colour of Torenia into white and pale (Guo et al., 2016).Suppression of chalcone isomerase (CHI) through RNAi silencing changes flavonoid components in tobacco flowers and reduces pigmentation (Guo Using dsRNA-CPP6 to target negative regulators of plant defence genes efficiently protected rice plants from bacterial infection.Plant-pathogenic bacteria can destroy whole crops if not controlled, they secrete effectors to hijack plant mechanisms to cause virulence (Ramu, Oh, et al., 2021).Many negative regulators are upregulated during pathogen infections along with effector-hijacked genes (Pal et al., 2022).Much evidence has been reported on possible target genes for virus, insect and fungal resistance (Rank & Koch, 2021); however, no study has reported targeting plant genes to improve bacterial disease resistance.Naked dsRNAs targeting different CYP51 genes in barley can reduce Fusarium wilt (Koch et al., 2019).dsRNA targeting sorbitol dehydrogenase and phospholipase D in potatoes reduced the sporulation of Phytophthora infestans (Kalyandurg et al., 2021).RNAimediated silencing has been used to improve the host-defence system in crop plants (Hollomon, 2012).In Agrobacterium tumefaciens infection, iaaM and ipt genes are involved in crown gall disease, and RNAi-mediated silencing reduces tumour production in Arabidopsis (Escobar et al., 2001).In legumes, several miRNA families target NBS-LRR receptors of plant innate immunity in tomatoes and other crops (Shivaprasad et al., 2012).
Targeting SDIR1, an E3 ligase that is a negative regulator of The Xoo TALE protein target SWEET14 plays a negative role in plant defence during bacterial blight disease.CRISPR mutants targeting the promoter of SWEET14 show improved resistance in rice plants (Oliva et al., 2019).The SWEET14 off-target gene PCD1 showed a higher levels of transcripts, which could also contribute to improved tolerance (Verma et al., 2019).The results suggest that dsRNA with CPP6 does not induce any off-target gene effects.
The transient silencing of SWEET14 using sweet14-dsRNA-CPP6 showed reduced bacterial disease symptoms and multiplication rate and enhanced resistance.Even after 30 days sweet14-dsRNA-CPP6-sprayed plants showed better tolerance against bacterial disease than dsRNA-sprayed plants and completely recovered.This suggests that the slow release of dsRNAs by CPP6 in silencing the target gene for a longer duration could provide durable crop protection.Our study demonstrates that the CPP-based nanoformulations could stabilize different dsRNA constructs and effectively deliver them into plants.Such nanoformulations can also be used to manipulate plant genes involved in stress tolerance and crop protection to attain sustainability.

| Plant growth and bacterial strain
The A. thaliana (Col-0) plants were grown in a growth chamber (22°C and 50%-60% relative humidity) with a 16/8 h light/dark period.The rice TN1 seeds were soaked in water overnight and germinated on filter paper in a Petri plate.Germinated seedlings were transferred to pots and maintained for 45 days in the greenhouse (28°C and 50%-60% relative humidity) with a 16/8 h light/ dark photoperiod.The Xoo strain was cultured in nutrient broth (NB) in a shaking incubator at 28°C for 2 days.Nutrient broth was prepared using peptone 5 g, NaCl 5 g, beef extract 1.5 g and yeast extract 1.5 g (HiMedia) in 1 L of distilled water (pH 7-7.2).Nutrient agar (NA) was prepared using peptone 5 g, NaCl 5 g, beef extract 1.5 g, yeast extract 1.5 g and agar powder 15 g (Sigma) in 1 L of distilled water.
Briefly, 10 mL of solution containing MgCl 2 (3 mmol) and AlCl 3 (1 mmol) was added to 40 mL of 0.15 M NaOH solution under vigorous mixing.Pure LDH slurry was obtained through centrifugation followed by washing and then dispersed in deionized water, and autoclaved at 100°C for 16 h, resulting in homogenous suspension.

| NIR labelling and confocal imaging
CPP6 was labelled with Cy7 using a synthetic chemistry approach as described previously (Yavvari et al., 2019).The Cy7-CPP6 fluorophore was used to test the systemic movement of polymer in plants using NIR imaging.Cy5-dUTP (650 nm excitation, 670 nm emission) labelled gfp-dsRNA and Cy5-gfp-dsRNA-CPP6 were infiltrated to 3-week-old N. benthamiana plants, at 24 hpi signals were visualized in 40× oil immersion using a confocal laser-scanning microscope (TCS SP5; Leica).Similarly, Cy5-gfp-dsRNA and Cy5-gfp-dsRNA-CPP6 were added to the tubes, and after 24 h, signals were visualized in rice leaves using confocal microscopy.

| Nanopolymer uptake and systemic movement in plants
To test the systemic movement of polymer into plants through infiltration or root uptake, a Cy7-labelled CPP6 polymer was infiltrated in 3-week-old N. benthamiana and A. thaliana plants.After 24 and 48 h of infiltration, the whole plant was visualized under SPECTRUM In Vivo Imaging System (Perkin Elmer).In rice, the labelled polymer was added in the water, allowed to be taken up through roots and visualized after 24 and 48 h for signals.

| Toxicity of nanopolymers on rice growth and development
TN1 seeds were soaked overnight with four different concentrations (5, 25, 50 and 75 μg) of each polymer CPP1, CPP2, CPP4, CPP6 and CPP8.For each concentration, 20 seedlings were used for germination on wet filter paper.Shoot and root length were measured after 4 and 7 days of germination.The seeds grown in distilled water were used as a control.
The predicted off-target genes for each dsRNA were also identified from the pssRNAit tool.The primers were designed for that region and custom synthesized (Table S2).T7 promoter sequences were added to each 5′ end of the DNA template using PCR amplification.DNA template was purified and converted to dsRNA using an in vitro transcription kit (New England Biolabs).Residual DNA was removed from the transcription reaction using DNase I treatment, purified using phenol-chloroform precipitation and eluted in 30 μL of nuclease-free water.The dsRNA concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher).For labelled dsRNA synthesis, 0.5 μL of 10 mM dUTP (uridine triphosphate) labelled with Cy5 was added during the in vitro transcription reaction.

| Preparation of dsRNA-nanoconjugate and biochemical characterization
In vitro-transcribed dsRNA was incubated with CPP6 at a 1:10 ratio at room temperature for 30 min.We performed dynamic light scattering for biochemical characterization by measuring the hydrodynamic diameter (in nm) to recognize any size variation for up to 25 days for gfp-dsRNA-CPP6 and 15 days for pds-dsRNA-CPP6 using Malvern instrument Zetasizer Nanoseries, Nano-ZS90.ZP (mV) was measured to assess the surface charge/potential of the complex.

| Stability and release of dsRNA with nanoparticles in different conditions
The 500 ng of gfp-dsRNA and 5000 ng of CPP6 were conjugated and mixed in tubes and then incubated at 4, 37 and 48°C for 0 min, 2 h, 6 h, 12 h, 24 h, 48 h, 3 days, 4 days, 5 days and 10 days.For pHdependent stability, the conjugated gfp-dsRNA-CPP6 was incubated at pH 7, 5, and 3 for 0 min, 2 h, 6 h, 12 h, 24 h, 48 h, 3 days, 4 days, 5 days and 10 days.The samples were analysed on 2% agarose gel electrophoresis.To assess the RNase-mediated degradation of gfp-dsRNA-CPP6 complexes, 1 μL (10 mg/mL) of RNase A (Thermo Fisher) was added to the complexes and incubated at 4, 37 and 48°C for 0, 30 min, 2 h, 6 h, 12 h, 24 h and 48 h and resolved on 2% agarose gel.For the release of dsRNA from the complex, different SDS concentrations were added in a reaction for 30 min and analysed on a 2% agarose gel.

| Transcription and translation assay
To check the DNA template accessibility for polymerase activity in the presence of CPP6, 50 ng template was used for PCR amplification, using GFP-specific primers in all reactions.CPP6 and gfp-dsRNA were added at 100 or 200 ng to the individual reactions, the gfp-dsRNA-CPP6 complex was made and added to the reaction.Similarly, combinations were made with LDH polymer, gfp-dsRNA, kept for 25 and 40 cycles in a thermal cycler and analysed on 0.8% agarose gel.For translation efficiency, dsRNA targeting luc was designed.The luc-dsRNA and luc-dsRNA-CPP6 were used in PURExpress In Vitro Protein Synthesis Kit (New England Biolabs) for assessing the translation.

| Arabidopsis GFP-expressing plants
To assess the gene silencing and monitor the expression pattern, RPL10-GFP transgenic plants were generated by the floral dip method.gfp-dsRNA-CPP6 complex was made by mixing in vitro-transcribed gfp-dsRNA with CPP6 at a 1:10 ratio.Naked gfp-dsRNA and gfp-dsRNA-CPP6 were infiltrated into the leaves of 3-week-old Arabidopsis RPL10-GFP-OE transgenic plants.GFP fluorescent signals were visualized under confocal microscopy at 1, 24, 48 hpi and leaf samples were collected for gene expression and western blot analysis.To ascertain the levels of GFP, total protein was extracted in extraction buffer (50 mM Tris MES pH 8.0, 50 mM NaCl, 1% vol/vol NP-40, 0.1%

| 3 of 15 PAL
et al. polymer (CPP) could effectively encapsulate dsRNA at lower concentrations than other reported polymers (Das & Sherif, 2020).CPP6 facilitated systemic spread of dsRNAs in plants following infiltration, foliar spray and root uptake.Arabidopsis plants overexpressing GFP were targeted by dsRNA and showed prolonged suppression of GFP fluorescence.The flowering-associated genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) in Arabidopsis were targeted by SIGS.Phytoene desaturase (PDS) and bZIP23 transcription factor were targeted through root uptake in rice.Foliar SIGS targeted against OsSWEET14 and OsSDIR1 provided resistance against Xoo infection.CPP6 nanopolymers could effectively deliver dsRNAs into the plants and prolonged silencing of the target gene through RNAi machinery activation.A low amount of dsRNA could successfully provide durable resistance against Pst and Xoo.
,b).Near-infrared (NIR) signals were visible in distal leaves, indicating the systemic movement of the polymer in plants.The uptake of the polymer through roots was assessed in rice seedlings; NIR signals were visible in the top leaves after 24 and 48 h (Figure2c).To visualize dsRNA uptake in plants, Cy5-labelled naked pds-dsRNA and Cy5-pds-dsRNA-CPP6 was infiltrated in N. benthamiana leaves; signals were detected 24 h post-infiltration (hpi) in plant cells (Figure2d).Similarly, rice seedlings were immersed in Cy5-dsRNA and Cy5-dsRNA-CPP6; fluorescence in the rice sheath at 24 hpi indicated uptake of labelled dsRNA through the roots (Figure2e).

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Cationic poly-aspartic acid-derived polymers (CPP) provide double-stranded RNA (dsRNA) stability.(a) Structure of different CPPs.Polymer-gfp-dsRNA complex formation with (b) CPP6 and, (c) layered double hydroxide (LDH) nanoparticles.Five hundred nanograms of in vitro-synthesized GFP-dsRNA was incubated for 30 min at room temperature with different ratios of polymers and loaded on 2% agarose gel.Naked gfp-dsRNA is visible in the gel whereas gfp-dsRNA-CPP6 and gfp-dsRNA-LDH complex remain in the wells.Hydrodynamic diameter size (nm) of (d) gfp-dsRNA-CPP6 and (e) pds-dsRNA-CPP6 complexes was measured after incubating dsRNA with polymer for 30 min at room temperature.(f) The zeta potential (ZP) of dsRNA-CPP6 complexes of GFP and PDS fragments.(g) Release of gfp-dsRNA using SDS from polymer complexes.The gfp-dsRNA-CPP6 complexes were treated with different concentrations of SDS for 30 min and resolved on 2% agarose gel.+ indicates 5 μg of CPP6 and 500 ng of dsRNA in each reaction.The big bright bands are higher concentrations of SDS forming micelles with ethidium bromide, giving fluorescent anionic bands that migrate down the gel.Stability of dsRNA and gfp-dsRNA-CPP6 complexes, (h) at different temperatures, (i) different pH and, (j) with RNase A treatment.Efficiency in interfering with DNA accessibility for amplification by (k) CPP6 and (l) LDH nanoparticles.In a PCR, different combinations of gfp-dsRNA were complexed with CPP6, LDH nanoparticle and added to the PCR with gfp template.(m) In vitro translation efficiency in the presence of CPP6, LDH nanoparticle with luc-dsRNA.luc-dsRNA and CPP6, complexes in the reaction mixture.Error bars indicate values of means ± SE from three biological replicates, experiments repeated two times with similar results.The significance of differences was examined using Student's t test (⍺ = 0.05, ****p < 0.0001).In the naked gfp-dsRNA-infiltrated plants, signals were reduced at 1 hpi; however, by 24 hpi the GFP signals had recovered.The gfp-dsRNA-CPP6-infiltrated plants showed significantly reduced GFP fluorescence even at 48 hpi (Figure 3c).The effect of dsRNA was assessed at the protein level using GFP-specific antibodies by western blotting at different time points.At 1 hpi, in gfp-dsRNA-and gfp-dsRNA-CPP6-infiltrated plants, GFP levels were reduced drastically compared to the GFP-OE control plants.At 24 hpi, GFP levels recovered to normal in naked dsRNA-infiltrated plants, whereas gfp-dsRNA-CPP6-infiltrated plants showed reduced GFP levels even at 48 hpi, suggesting CPP6 could provide prolonged effectiveness for gene silencing.The GFP in distal leaves of gfp-dsRNA-CPP6-infiltrated F I G U R E 2 CPP6 facilitates double-stranded RNA (dsRNA) uptake in plants.In planta uptake and translocation of CPP6 polymer.The Cy7-fluorophore was attached to CPP6 and infiltrated into (a) Nicotiana benthamiana and (b) Arabidopsis then visualized using a near-infrared (NIR) imaging system at 24 and 48 h post-infiltration (hpi).(c) Uptake of CPP6-Cy7 through roots by rice seedlings.NIR signals were visualized at 24 and 48 hpi.Visualization of pds-dsRNA in plant leaves by confocal microscopy.pds-dsRNA labelled with Cy5-dUTP, Cy5-dUTP-dsRNA-CPP6 complexes, (d) in N. benthamiana at 24 hpi and (e) uptake through roots in rice at 24 hpi.F I G U R E 3 Silencing of green fluorescent protein (GFP) transgene in Arabidopsis using gfp-dsRNA.(a) Schematic representation of the application of gfp-dsRNA-CPP6 through syringe infiltration in transgenic RPL10-GFP-OE Arabidopsis plants.Three-week-old plants were infiltrated with gfp-dsRNA or gfp-dsRNA-CPP6, control plants were mock-treated with water.(b) GFP transcript levels in gfp-dsRNA-and gfp-dsRNA-CPP6-treated plants at 1, 24 and 48 h post-infiltration (hpi).(c) Confocal microscopic images showing GFP fluorescence signals in gfp-dsRNA-and gfp-dsRNA-CPP6-infiltrated leaves at 1, 24 and 48 hpi.(d) Immunoblot (IB) showing RPL10-GFP levels in leaves at 1, 24 and 48 hpi from the infiltration site and in distal leaves.The total protein was isolated from the leaves, and equal concentrations were loaded for each sample on SDS-PAGE.Anti-GFP antibody was used to detect the levels of protein.Ponceau S staining was done to analyse the protein normalization.Error bars indicate values of means ± SE from three biological replicates.The significance of differences was examined using Student's t test (⍺ = 0.05, ***p < 0.001).PAL et al.
48 h post-spraying (hps), fewer plants sprayed with naked ft-dsRNA or ft-dsRNA-CPP6 showed early bolting initiation compared to water-and CPP6-sprayed plants (Figure4b,c).At 10 dps, bolting length was reduced >2-fold and >4-fold in ft-dsRNA-and ft-dsRNA-CPP6treated plants, respectively (Figure4d,e).At 2, 4, 6 and 10 dps, FT transcript levels were unchanged in leaves, whereas in floral parts at 10 dps transcript levels were reduced >2-fold and >4-fold in plants sprayed with ft-dsRNA and ft-dsRNA-CPP6, respectively (Figure4f).The FT protein levels were assessed in treated plants at different time points and reduction was observed at 4 dps and 10 dps in floral parts of ft-dsRNA-CPP6-sprayed plants compared to water-and CPP6sprayed plants.In the naked ft-dsRNA-treated plants, the reduction was observed only in floral parts at 10 dps (Figure4g).The predicted off-target gene Magnesium transporter (MgT) levels were reduced in ft-dsRNA-treated plants (Figure4h), which may reflect a role in floral bud development and fertility(Pabón-Mora et al., 2022).Delayed flowering of the dsRNA-treated plants does not signal the induction of MgT and thus less transcript levels were observed (FigureS3).

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Delayed flowering in Arabidopsis Col-0 plants targeting flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) through foliar application of double-stranded RNA (dsRNA).(a) Schematic representation of FT and PIF4 role in flowering regulation and application of specific dsRNA targeting these genes to delay the flowering.dsRNA concentration of 125 ng/plant with CPP6 was sprayed before bolting and phenotypic characters were observed.Plants sprayed with water or CPP6 were used as the controls.A minimum of 20 plants for each treatment was used.(b) Phenotype of delayed bolting initiation in plants sprayed with ft-dsRNA and ft-dsRNA-CPP6 complex at 48 h post-spraying (hps).(c) Percentage of plants showing bolting initiation in ft-dsRNA-and ft-dsRNA-CPP6-sprayed plants at 48 hps.Error bars indicate the average percentage from three experiments with a minimum of 20 plants.(d) Phenotype of plants sprayed with ft-dsRNA or ft-dsRNA-CPP6 compared to water-and CPP6-sprayed plants after 10 days post-spraying (dps).(e) Bolting length in ft-dsRNA-and ft-dsRNA-CPP6-sprayed plants at 10 dps.(f) Expression of FT in leaf samples collected after 2, 4, 6, 10 dps and floral parts at 10 dps compared to water-and CPP6-sprayed plants.(g) FT levels at different time intervals were assessed by immunoblotting using FT-specific antibodies.(h) Expression of predicted off-target genes Magnesium transporter (MgT) and (i) Pectin methyl transferase inhibitor (PMEI) at 10 dps in floral parts, (j) phenotype of PIF4 targeted plants sprayed with water, CPP6, pif4-dsRNA or pif4-dsRNA-CPP6 at 7 dps.(k) Bolting length at 10 dps in plants sprayed with pif4-dsRNA, pif4-dsRNA-CPP6 or water.(l) Expression of PIF4 in leaf samples collected at 2, 4, 6, 10 dps and floral parts at 10 dps compared to water-and CPP6-sprayed plants.Error bars indicate values of means ± SE from three biological replicates.A minimum of 24 plants was used with similar results repeated three times.The significance of differences were examined using Student's t test (⍺ = 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).transcription of SDIR1 similar to PDS (Figure S4b).The activation of the silencing machinery was assessed by quantifying the expression of Dicer-like (DICER) and Argonaute (AGO12) expression.Expression of AGO12 and DICER1 were upregulated >2-fold in sdir1-dsRNA-and sdir1-dsRNA-CPP6-treated plants, suggesting that the RNAi machinery was activated in plants with a disease-resistance phenotype (Figure 6i,j).Xoo uses the type III secretion system to release TALEs into the host cell, which bind to the promoter of the Sucrose Will be Eventually Exported Transporter (SWEET14) gene.SWEET14 transporter exports sucrose to the apoplastic region, which favours bacterial survival and multiplication.SWEET14 acts as a negative regulator of plant defence; therefore, transiently silencing it through dsRNA during pathogen infection could protect rice against BLB disease.The 45-day-old TN1 rice plants were infected with Xoo then after 24 hpi, sweet14-dsRNA (250 ng/leaf) and sweet14-dsRNA-CPP6 complexes were sprayed.Bacterial blight disease symptoms at 10 dps were reduced in sweet14-dsRNA-and sweet14-dsRNA-CPP6-sprayed plants compared to unsprayed control and CPP6-sprayed plants (Figure 7a).The bacterial growth at 2 dps was reduced >1-fold in sweet14-dsRNA-and >2-fold in sweet14-dsRNA-CPP6-sprayed plants (Figure 7b).Lesion length reduced >2-fold in sweet14-dsRNA-and >4-fold in sweet14-dsRNA-CPP6-sprayed plants (Figure 7c).The transcript levels of SWEET14 remained unchanged at 2 dps in sweet14-dsRNA-treated plants, whereas it was reduced >1-fold in sweet14-dsRNA-CPP6sprayed plants.At 4 dps, transcripts were reduced >3-fold in sweet14-dsRNA-sprayed plants and >6-fold in sweet14-dsRNA-CPP6-sprayed plants.At 6 dps SWEET14 transcripts were reduced >9 fold in sweet14-dsRNA-and sweet14-dsRNA-CPP6-sprayed F I G U R E 5 Effectiveness of dsRNA-CPP6 targeting phytoene desaturase synthase (PDS) and OsbZIP23 through root uptake in rice.(a) The phenotype of rice seedlings showing stunted, yellowing and albino leaves in pds-dsRNA and pds-dsRNA-CPP6 treatments.Rice seedlings grown for 3 days in the light were transferred to microcentrifuge tubes in water.Two micrograms of pds-dsRNA and pds-dsRNA-CPP6 was added to individual vials and kept in the dark for 10 days.Plants treated with water or CPP6 were used as controls.(b) Seedling height in pds-dsRNA-and pds-dsRNA-CPP6-treated plants compared to treatment with water and CPP6 10 days after treatment.(c) PDS transcript levels 48 h after treatment.Total RNA from leaf samples was isolated and converted to cDNA and used as template.(d) Phenotypic images of rice seedlings exposed to 150 mM NaCl stress, bzip23-dsRNA, bzip23-dsRNA-CPP6 and bzip23-dsRNA-CPP6 + NaCl.Photographs were taken after 48 h of exposure.(e) Shoot and root length of treated seedlings were recorded 48 h after treatment.(f) Expression of OsbZIP23 and (g) bZIP23 transcription factor target gene OTS1 in NaCl-and bzip23-dsRNA-treated samples.The pregerminated seedlings were treated with 150 mM NaCl for 24 h and then treated with 150 ng bzip23-dsRNA, bzip23-dsRNA-CPP6 and allowed to grow for 48 h.A minimum of 10 seedlings was used for each treatment.Error bars indicate values of means ± SE from three biological replicates.The significance of differences was examined by Student's t test (⍺ = 0.05, *p < 0.05, **p < 0.01).

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I G U R E 7 Double-stranded RNA (dsRNA) targeting the SWEET14 gene improved disease resistance against Xanthomonas oryzae pv.oryzae (Xoo).Rice TN1 plants (45 day old) were infected with 10 6 cfu/mL Xoo using the leaf-clipping method and 24 h post-inoculation (hpi), CPP6, sweet14-dsRNA or sweet14-dsRNA-CPP6 was sprayed on plants.(a) Bacterial blight disease symptoms at 10 days post-spraying (dps) in leaves sprayed with sweet14-dsRNA or sweet14-dsRNA-CPP6.(b) Bacterial multiplication rate, (c) lesion length at 10 dps and (d) expression of SWEET14 in plants sprayed with sweet14-dsRNA and sweet14-dsRNA-CPP6 compared to water-and CPP6-sprayed plants.(e) Expression of predicted off-target gene PCD at 2, 4, 6 and 10 dps.(f) Prolonged survival of sweet14-dsRNA-CPP6-sprayed plants from bacterial infection even after 30 dps, photographs were taken 60 days after germination.Error bars indicate values of means ± SE from three biological replicates.The significance of differences was exmined using Student's t test (⍺ = 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).| 11 of 15 PAL et al. et al., 2016).The exogenous application of dsRNAs through CPP6 targeting phytoene desaturase (OsPDS1) leads to stunted growth and yellowing of the rice leaves due to the reduced accumulation of carotenoids.Seedlings attained this phenotype by taking up the pds-dsRNA through roots and CPP6 could be systemically translocated to shoots to induce silencing of the PDS gene.
plant defence, provided protection against bacterial leaf blight caused by Xoo in rice plants.Targeting SDIR1 inhibited the bacterial multiplication rate and reduced lesion lengths in dsRNA-CPP6-sprayed plants.Induction of Argonaute and Dicer-like protein 1 indicates activation of the RNAi machinery in sdir1-dsRNAsprayed plants.The induction of these genes in dsRNA-CPP6 is lower because of the slow release of dsRNA in the plant system.